Methods for treating or preventing angiogenesis-dependent symptoms

ABSTRACT

A method for enhancing the transfection efficiency of naked plasmid DNA in treating and/or preventing angiogenesis-dependent symptoms is provided by the present inventions. According to the present method, a suitable naked plasmid DNA is subjected for intramuscular injection under increased pressure inside the muscle or hyperbaric oxygen. Angiogenesis-dependent symptoms, including wounds, inflammatory diseases, critical limb ischemia, ischemic heart diseases, cerebral infarction, diabetic neuropathy, spinal canal stenosis, etc., may be treated by the present methods.

TECHNICAL FIELD

The present invention relates to methods for treating and/or preventingangiogenesis-dependent symptoms by administering an intramuscularinjection of a naked plasmid DNA under specific conditions, such asincreased pressure inside the muscle or hyperbaric oxygen.

BACKGROUND ART

Recent progress in molecular biology has led to the development of genetherapy as a new treatment strategy for cardiovascular diseases.Targeted diseases range from single gene deficiency diseases to morecomplex diseases in adults, such as peripheral arterial diseases. Forexample, critical limb ischemia is estimated to develop in 500 to 1000per million individuals in one year (“Second European Consensus Documenton Chronic Critical Leg Ischemia.”, Circulation 84(4 Suppl.) IV 1-26(1991)). In patients with critical limb ischemia, amputation, despiteits associated morbidity, mortality and functional implications, isoften recommended as a solution against disabling symptoms (M. R.Tyrrell et al., Br. J. Surg. 80: 177-180 (1993); M. Eneroth et al., Int.Orthop. 16: 383-387 (1992)). There exists no optimal medical therapy forcritical limb ischemia (Circulation 84(4 Suppl.): IV 1-26 (1991)).

Recently, the efficacy of therapeutic angiogenesis by gene transfer ofvascular endothelial growth factor (VEGF) has been reported to beeffective for human patients with critical limb ischemia (I. Baumgartneret al., Circulation 97: 1114-1123 (1998); J. M. Isner et al., J. Vasc.Surg. 28: 964-973 (1998); I. Baumgartner et al., Ann. Intern. Med. 132:880-884 (2000)) and myocardial ischemia (D. W. Losordo et al.,Circulation 98: 2800-2804 (1998); P. R. Vale et al., Circulation 102:965-974 (2000); T. K. Rosengart et al., Circulation 100: 468-474 (1999);T. K. Rosengart et al., Ann. Surg. 230: 466-470 (1999)). In addition toVEGF, gene transfer of other angiogenic growth factors, includingfibroblast growth factor (FGF), hepatocyte growth factor (HGF) andhypoxia-inducible factor (HIF), has also been reported to stimulatecollateral formation (Y. Taniyama et al., Gene Ther. 8: 181-189 (2000);H. Tabata et al., Cardiovasc. Res. 35: 470-479 (1997); H. Ueno et al.,Arterioscler. Thromb. Vasc. Biol. 17: 2453-2460 (1997); K. A. Vincent etal., Circulation 102: 2255-2261 (2000); F. J. Giordano et al., Nat. Med.2: 534-539 (1996); M. Aoki et al., Gene Ther. 7: 417-427 (2000); H. Uedaet al., Ann. Thorac. Surg. 67: 1726-1731 (1999); E. R. Schwarz et al.,J. Am. Coll. Cardiol. 35: 1323-1330 (2000)).

The feasibility of gene therapy using angiogenic growth factors to treatperipheral arterial disease seems to be superior to recombinant proteintherapy. For example, through gene therapy, one can potentially maintainan optimally high and local concentration over time. Thus, in the caseof therapeutic angiogenesis, to avoid side effects, it may be desirableto deliver a lower dose of protein through an actively expressedtransgene in the artery over a period of several days or more, ratherthan administering a single or multiple bolus doses of recombinantprotein. Interestingly, most successful clinical trials treatingperipheral arterial diseases using angiogenic growth factors haveinvolved intramuscular transfection of naked plasmid DNA (I. Baumgartneret al., Circulation 97: 1114-1123 (1998); J. M. Isner et al., J. Vasc.Surg. 28: 964-973 (1998); I. Baumgartner et al., Ann. Intern. Med. 132:880-884 (2000); D. W. Losordo et al., Circulation 98: 2800-2804 (1998);P. R. Vale et al., Circulation 102: 965-974 (2000)). However, such invivo gene transfer, by direct injection of “naked” plasmid DNA intoskeletal muscle, has been known to be inefficient.

Therefore, more efficient methods for gene transfer are required in theart for therapeutic application. Thus, many investigators have beenfocusing on alternate methods, such as the adenoviral gene transfermethod (H. Ueno et al. Arterioscler. Thromb. Vasc. Biol. 17: 2453-2460(1997); F. J. Giordano et al., Nat. Med. 2: 534-539 (1996); D. F.Lazarous et al., Cardiovasc. Res. 44: 294-302 (1999); L. Y. Lee et al.,Ann. Thorac. Surg. 69: 14-23 (2000); L. H. Gowdak et al., Circulation102: 565-571 (2000); O. Varenne et al., Hum. Gene Ther. 10:1105-1115(1999); E. Barr et al., Gene Ther. 1: 51-58 (1994)). Although adenoviralvectors are efficient (H. Ueno et al., Arterioscler. Thromb. Vasc. Biol.17: 2453-2460 (1997); F. J. Giordano et al., Nat. Med. 2: 534-539(1996); D. F. Lazarous et al., Cardiovasc. Res. 44: 294-302 (1999); L.Y. Lee et al., Ann. Thorac. Surg. 69: 14-23 (2000); L. H. Gowdak et al.,Circulation 102: 565-571 (2000); O. Varenne et al., Hum. Gene Ther.10:1105-1115 (1999); E. Barr et al., Gene Ther. 1: 51-58 (1994)), theyhave some theoretical disadvantages, such as induction of strongimmunogenicity in the host (V. J. Dzau et al., Proc. Natl. Acad. Sci.USA 93: 11421-11425 (1996)). In addition to efficiency, safety is alsoan important issue for gene transfer methods. The infusion of adenovirushas recently been reported to cause deleterious side effects (E.Marshall, Science 286: 2244-2245 (1999)). Thus, in the interests ofsafety, it would be more desirable to make non-virus-mediated plasmidDNA more efficient to achieve an ideal treatment for peripheral arterialdiseases. Such innovation in plasmid DNA-based gene transfer shouldprovide methods with high transfection efficiency without severe sideeffects.

To increase the transfection efficiency of naked plasmid DNA, thepresent inventors previously tested the use of ultrasound and echocontrast microbubbles (Optison® (FS069); Molecular Biosystems). As aresult, the inventors discovered that high transfection efficiency couldbe achieved by ultrasound-mediated plasmid DNA transfection using echocontrast microbubbles (Y. Taniyama et al., Circulation 105: 1233-1239(2002); Y. Taniyama et al., Gene Therapy 9: 372-380 (2002)). Usingultrasound exposure in the presence of microbubble echo contrast agents,approximately 300-fold increment in transgene expression following nakedDNA transfection was reported in in vitro experiments (A. Lawrie et al.,Gene Ther. 9: 372-380 (2002)) In addition, the inventors confirmed theusefulness of ultrasound-mediated plasmid DNA transfection with Optison®into rat skeletal muscle as well as rat carotid artery (Y. Taniyama etal., Circulation 105: 1233-1239 (2002); Y. Taniyama et al., Gene Therapy9: 372-380 (2002)). Due to the appearance of transient holes in the cellmembrane through the spreading of the bubbles, this method increased thetransfection efficiency.

DISCLOSURE OF THE INVENTION

Although clinical trials of stimulation of angiogenesis by transfectionof angiogenic growth factors via intramuscular injection of nakedplasmid DNA have been successful, there still are unresolved problems inhuman gene therapy, including low transfection efficiency and safety.From this viewpoint, methods that achieve higher transfection efficiencyfor naked plasmid DNA are desired in the art.

The object of the present invention is to provide methods for treatingand/or preventing angiogenesis-dependent symptoms by administering nakedplasmid DNA with high transfection efficiency. As described above, thepresent inventors previously investigated the use of ultrasound-mediatedplasmid DNA transfection using echo contrast microbubbles. Based on theefficient transfection achieved by this method, the inventors thoughtthat destabilizes the cellular membrane through high osmotic pressuremight increase the transfection efficiency of the naked plasmid DNAmethod. Therefore, the present inventors examined various agents andpressures on the injected site for their effect on the transfectionefficiency of naked plasmid DNA in vivo.

First, the present inventors examined the effects of injection volume onthe efficiency of naked plasmid DNA transfection into the cells of theskeletal muscles. Luciferase plasmid DNA dissolved in various volumes ofsolvent was subjected for intramuscular injection into the rat hindlimb.According to the present data, the transfection efficiency of nakedplasmid DNA seemed to be determined by the amounts of plasmid DNA aswell as the injection volume of solution injected into the skeletalmuscle, a phenomenon seemingly caused by the increase in pressure on thecellular surface. However, applying pressure to the site of injectionfrom outside the body (using the manchette of a sphygmomanometer on thehindlimb, for example) did not increase the transfection efficiency. Incontrast, injection of phosphate-buffered saline (PBS) solution 30minutes after plasmid DNA injection increased the transfectionefficiency of the plasmid DNA, whereas additional injection of PBSsolution after 5 hours did not. These data clearly demonstrate that highpressure inside the muscle is critical for increasing transfectionefficiency.

Furthermore, the present inventors discovered that the intramuscularinjection of plasmid DNA in combination with hyperbaric oxygen (HBO)therapy enhances the transfection efficiency of naked plasmid DNA.Moreover, the influence of the kind of solutions for dissolving theplasmid DNA was also determined. High transfection efficiency wasachieved by saline as well as PBS, but not with water. Interestingly,sucrose solution rather than glucose solution resulted in highluciferase activity.

Overall, the transfection efficiency of intramuscular injection ofplasmid DNA was enhanced by increases in the injection volume andosmotic pressure. Gene therapy using naked plasmid DNA of angiogenicgrowth factors with HBO therapy may provide a safe clinical gene therapyfor arterial diseases without viral vector.

Thus, the present invention provides methods for treating and/orpreventing angiogenesis-dependent symptoms by administering nakedplasmid DNA under increased pressure at the administration site or incombination with HBO therapy. More specifically, the present inventionprovides:

(1) a method for treating or preventing an angiogenesis-dependentsymptom comprising the step of injecting a suitable naked plasmid DNAinto a muscle under a condition wherein the pressure inside the muscleis increased;

(2) the method of (1), wherein the pressure inside the muscle isincreased by adopting a large injection volume;

(3) the method of (1), wherein the pressure inside the muscle isincreased by injecting PBS after plasmid DNA administration;

(4) the method of (1), wherein the naked plasmid DNA is diluted insaline, PBS, sucrose solution, or glucose solution;

(5) the method of (1), wherein the naked plasmid DNA encodes anangiogenic growth factor;

(6) the method of (5), wherein the angiogenic growth factor is selectedfrom the group consisting of: hepatocyte growth factor (HGF); vascularendothelial growth factor (VEGF); fibroblast growth factor (FGF); andnitric oxide synthase, including macrophage derived nitric oxidesynthase, inducible nitric oxide synthase, and brain derived nitricoxide synthase;

(7) the method of (1), wherein the angiogenesis-dependent symptom isselected from the group consisting of: wounds, including bedsore andskin ulcer; inflammatory diseases; critical limb ischemia; ischemicheart diseases, such as myocardial infarction, angina pectoris, andheart failure; cerebral infarction; diabetic neuropathy; and spinalcanal stenosis;

(8) a method for treating or preventing angiogenesis-dependent symptomthrough intramuscular injection of naked plasmid DNA in combination withhyperbaric oxygen (HBO) therapy;

(9) the method of (8), wherein the HBO therapy is conducted by exposureof 100% oxygen;

(10) the method of (8), wherein the subject to be treated is subjectedto the HBO therapy immediately after plasmid DNA administration;

(11) the method of (8), wherein the naked plasmid DNA is diluted insaline, PBS, sucrose solution, or glucose solution;

(12) the method of (8), wherein the naked plasmid DNA encodes anangiogenic growth factor;

(13) the method of (12), wherein the angiogenic growth factor isselected from the group consisting of: hepatocyte growth factor (HGF);vascular endothelial growth factor (VEGF); fibroblast growth factor(FGF); and nitric oxide synthase, including macrophage derived nitricoxide synthase, inducible nitric oxide synthase, and brain derivednitric oxide synthase; and

(14) the method of (8), wherein the angiogenesis-dependent symptom isselected from the group consisting of: wounds, including bedsore andskin ulcer; inflammatory diseases; critical limb ischemia; ischemicheart diseases, such as myocardial infarction, angina pectoris, andheart failure; cerebral infarction; diabetic neuropathy; and spinalcanal stenosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph showing the comparison of luciferase activitiesdetected 2 days after transfection of naked plasmid DNA at variousinjection volumes into the skeletal muscle in vivo. Naked luciferaseplasmid DNA (200 or 800 μg) diluted in 100, 200, 300 and 400 μl PBS weretransfected into rats. Each group contained 6 animals. **p<0.01 vs. 100μl.

FIG. 2 depicts a graph showing the comparison of luciferase activitiesdetected 2 days after transfection of naked plasmid DNA at variousinjection volumes and different sites of the skeletal muscle in vivo.200 ug/100×1: rats transfected with naked luciferase plasmid DNA (200μg) diluted in 100 μl PBS at one site; 200 ug/25×4: rats transfectedwith naked luciferase plasmid DNA (0.200 μg) diluted in 25 μl PBS at 4sites; 200 ug/12.5×8: rats transfected with naked luciferase plasmid DNA(200 μg) diluted in 12.5 μl PBS at 8 sites; and 400 ug/25×4: ratstransfected with naked luciferase plasmid DNA (400 μg) diluted in 25 μlPBS at 4 sites. Each group contained 10 animals. **p<0.01 vs. 200g/100×1.

FIG. 3 depicts a graph showing the comparison of luciferase activitiesdetected 2 days after transfection of naked plasmid DNA into theskeletal muscle using manchette wrapping. The manchette ofsphygmomanometer was wrapped on the muscle transfected with nakedluciferase plasmid DNA immediately after transfection. 200/100: ratstransfected with naked luciferase plasmid DNA (200 μg) diluted in 100 μlPBS without manchette wrapping; 10 s/10(300): rats transfected withnaked luciferase plasmid DNA (200 μg) diluted in 200 μl PBS compressed10 times by the manchette for 10 seconds at 300 mmHg; 10 s/30(300): ratstransfected with naked luciferase plasmid DNA (200 μg) diluted in 200 μlPBS compressed 30 times by the manchette for 10 seconds at 300 mmHg; 1m/10(150): rats transfected with naked luciferase plasmid DNA (200 μg)diluted in 200 μl PBS compressed 10 times by the manchette for 1 minuteat 150 mmHg; 1 m/10(300): rats transfected with naked luciferase plasmidDNA (200 μg) diluted in 200 μl PBS compressed 10 times by the cuff for 1minute at 300 mmHg; 5 m/1(150): rats transfected with naked luciferaseplasmid DNA (200 μg) diluted in 200 μl PBS compressed 1 time by themanchette for 5 minutes at 150 mmHg; and 5 m/1(300) rats transfectedwith naked luciferase plasmid DNA (200 μg) diluted in 200 μl PBScompressed 1 time by the manchette for 5 minutes at 300 mmHg. Each groupcontained 4 animals. *p<0.05 vs. 200/100.

FIG. 4 a depicts a graph showing the comparison of luciferase activitiesdetected 2 days after transfection of naked plasmid DNA (200 μg) intothe skeletal muscle with additional injection of PBS solution. A PBSsolution without plasmid DNA was additionally injected intramuscularlyafter 0.5 or 5 hours after the transfection of naked luciferase plasmidDNA into the same site of the muscle. 200 ug/100: rats transfected withnaked luciferase plasmid DNA (200 μg) diluted in 100 μl PBS; 200ug/100+300(0.5 h): rats transfected with naked luciferase plasmid DNA(200 μg) diluted in 100 μl PBS followed by injection of 300 μl PBS 30minutes after the transfection; 200 ug/100+300(5 h): rats transfectedwith naked luciferase plasmid DNA (200 μg) diluted in 100 μl PBSfollowed by injection of 300 μl PBS 5 hours after the transfection; and200 ug/400: rats transfected with naked luciferase plasmid DNA (200 μg)diluted in 400 μl PBS. Each group contained 4 animals. **p<0.01 vs. 200ug/100.

FIG. 4 b depicts a graph showing the comparison of luciferase activitiesdetected 2 days after transfection of naked plasmid DNA (800 μg) intothe skeletal muscle with additional injection of PBS solution. A PBSsolution without plasmid DNA was additionally injected intramuscularlyafter 0.5 or 5 hours after the transfection of naked luciferase plasmidDNA to into the same site of the muscle. 800 ug/100: rats transfectedwith naked luciferase plasmid DNA (800 μg) diluted in 100 μl PBS; 800ug/100+300(0.5 h): rats transfected with naked luciferase plasmid DNA(800 μg) diluted in 100 μl PBS followed by injection of 300 μl PBS alone30 minutes after the transfection; 800 ug/100+300(5 h): rats transfectedwith naked luciferase plasmid DNA (800 μg) diluted in 100 μl PBSfollowed by injection of 300 μl PBS 5 hours after the transfection; and800 ug/400: rats transfected with naked luciferase plasmid DNA (800 μg)diluted in 400 μl PBS. Each group contained 4 animals. **p<0.01 vs. 800ug/100.

FIG. 5 depicts a graph showing the effect of HBO therapy on luciferaseactivity 2 days after transfection of naked plasmid DNA (200 μg) intothe skeletal muscle. 200 ug/100: rats transfected with naked luciferaseplasmid DNA (200 μg) diluted in 100 μl PBS; and 200 ug/300: ratstransfected with naked luciferase plasmid DNA (200 μg) diluted in 300 μlPBS. normal: normal condition; HBO: HBO therapy with 100% O₂ at 2 atmfor 1 hour. Each group contained 3 animals. **p<0.01 vs. 200 ug/100.

FIG. 6 a depicts a graph showing effects of various solutions on theluciferase activity 2 days after the transfection of naked plasmid DNA(200 μg) into the skeletal muscle. Intramuscular injection of luciferaseplasmid DNA (200 μg) diluted in various solutions (200 μl injectionvolume) was performed. PBS: phosphate buffer saline; BSS: balancedsaline solution; and TE: Tris-HCl EDTA buffer. Each group contained 6animals. **p<0.01 vs. water.

FIG. 6 b depicts a graph showing the effects of glucose and sucroseconcentration on the luciferase activity 2 days after transfection ofnaked plasmid DNA (200 μg) into the skeletal muscle. Intramuscularinjection of luciferase plasmid DNA (200 μg) using glucose (5, 20 and50%) or sucrose (10, 30 and 50%) comprising solutions (200 μl injectionvolume) was performed. Each group contained 6 animals. **p<0.01 vs.water.

BEST MODE FOR CARRYING OUT THE INVENTION

The words “a”, “an” and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

To find an optimal condition for plasmid DNA transfer into skeletalmuscle, the present inventors modified the plasmid DNA gene transfermethod. First, the inventors examined the influence of injection volumeof plasmid DNA solution on the transfection efficiency. Next, theinfluence of the kind of solution to dissolve plasmid DNA was examined.Furthermore, the combined application of hyperbaric oxygen (HBO) therapywith plasmid DNA transfer was examined.

As a result, the transfection efficiency of naked plasmid DNA appearedto be increased by high pressure inside the muscle. Thus, the presentinvention provides a method for treating or preventingangiogenesis-dependent symptoms by intramuscular injection of a suitablenaked plasmid DNA under a condition wherein the pressure inside themuscle is increased. The invention provides a method for alleviating anangiogenesis-dependent symptom, inhibiting development of the symptom,or suppressing the symptom in a subject.

According to the present invention, the phrase “angiogenesis-dependentsymptoms” refers to symptoms of diseases that can be prevented,alleviated, improved or treated by angiogenesis. The symptoms that canbe treated or prevented according to the present invention include:wounds, including bedsores and skin ulcers; inflammatory diseases;critical limb ischemia; ischemic heart diseases, such as myocardialinfarction, angina pectoris and heart failure; cerebral infarction;diabetic neuropathy; and spinal canal stenosis.

Any plasmid DNA may be used for the present invention so long as theplasmid DNA contains a gene that encodes an angiogenic growth factor inan expressible manner upon introduction into the host. The gene encodingan angiogenic growth factor of the present invention is not limited inany way and includes those encoding proteins, polypeptides and partsthereof, so long as it has the ability to alleviate, improve or suppressthe angiogenesis-dependent symptoms or prevents the development of thesymptoms. Examples of preferred genes of the present invention include,but are not limited to, those encoding hepatocyte growth factor (HGF);vascular endothelial growth factor (VEGF); fibroblast growth factors(FGF), such as acidic FGF, basic FGF and FGF-4; nitric oxide synthases(NOS); VEGF-2; transforming growth factor (TGF)-α; TGF-β;platelet-derived (PD)-endothelial cell growth factor (ECGF);platelet-derived growth factor (PDGF); tumor necrosis factor (TNF)-α;insulin-like growth factor and antiopoietin-1.

The nucleotide sequence of a gene encoding HGF is described in theliterature (Nature 342: 440 (1989); Japanese Patent No. 2577091;Biochem. Biophys. Res. Commun. 163: 967 (1989); Biochem. Biophys. Res.Commun. 172: 321 (1990)). Any of these disclosed sequences may be usedas the angiogenic growth factor-encoding gene in the present invention.

Four subtypes are reported for the VEGF gene (VEGF121, VEGF165, VEGF189and VEGF206; Science 219: 983 (1983); J. Clin. Invest. 84: 1470 (1989);Biochem. Biophys. Res. Commun. 161: 851 (1989)). Any one of them may beused as the angiogenic growth factor-encoding gene in the presentinvention. However, among the four, VEGF165 is known to possess thestrongest biological activity, and thus is more preferred in the presentinvention.

Several isoforms of NOS have been isolated, including NOS isolated from:brain (nNOS; Bredt and Snyder, Proc. Natl. Acad. Sci. USA 87: 682-685(1990)); endothelial cells (eNOS; Fostermann et al., Biochem. Pharmacol.42: 1849-1857 (1991)); macrophages (iNOS; Hibbs et al., Science 235: 473(1987); Stuehr et al., Proc. Natl. Acad. Sci. USA 88: 7773-7777 (1991));hepatocytes (Knowles et al., Biochem. J. 279: 833-836 (1990)); vascularcells (Wood et al., Biochem. Biophys. Res. Commun. 170: 80-88 (1991);and neutrophils (Yui et al., J. Biol. Chem. 266: 12544-12547 (1991); Yuiet al., J. Biol. Chem. 266: 3369-3371 (1991)). In addition, NOS has beenalso isolated from other tissues (see, e.g., Hevel et al., J. Biol.Chem. 266: 22789-22791 (1991); Ohshima et al., Biochem. Biophys. Res.Commun. 183: 238-244 (1992); Hiki et al., J. Biochem. 111: 556-558(1992); Evans et al., Proc. Natl. Acad. Sci. USA 89: 5361-5365 (1992);Sherman et al., Biochemistry 32: 11600-11605 (1993)). Thus, genesencoding the above-mentioned NOS derived from various organs and tissuescan be used as the gene encoding angiogenic growth factor of the presentinvention. For example, the nucleotide sequence and amino acid sequenceof human eNOS is publicly available through the GenBank database(GenBank Accession Nos. AF400594 and P29474, respectively; see alsoJanssens et al., J. Biol. Chem. 267(21): 14511-14522 (1992); Marsden etal., FEBS Lett. 307(3): 287-293 (1992)). In addition, isoforms are knownto exist for eNOS (Fischman et al., Nat. Struct. Biol. 6(3): 233-242(1999)); such isoforms are also included in the NOS of the presentinvention. Further sequence information of NOS that can be used in thepresent invention include those of mammalian calmodulin-dependent NOS(nNOS; U.S. Pat. No. 5,268,465), human inducible NOS (iNOS; U.S. Pat.No. 5,468,630) and bovine endothelial NOS (eNOS; U.S. Pat. No.5,498,539).

Those skilled in the art can obtain cDNA that encodes an angiogenicgrowth factor by, for example, reverse transcriptase polymerase chainreaction (RT-PCR), using primers constructed from the publicly availablesequence information for the above-mentioned genes (see, e.g., MolecularCloning 2 nd ed., Cold Spring Harbor Laboratory Press (1989); PCR: aPractical Approach, IRL Press, Oxford (1991)) from sources comprisingthe angiogenic growth factor-encoding gene, which include cDNA librariesand genomic libraries of any mammalian species. However, in terms ofimmunogenicity, it is preferable to use genes from the same source asthe animal to be treated with the gene.

The gene encoding an angiogenic growth factor used in the presentinvention is not limited to those described above. Rather, a gene issuitable for the present invention so long as it codes for a proteinhaving angiogenic activity and includes: (1) a nucleotide sequence thathybridizes under stringent conditions to one of the above-describedcDNA; and (2) a nucleotide sequence encoding a protein comprising theamino acid sequence encoded by the above-mentioned cDNA, in which one ormore amino acids are substituted, deleted, added and/or inserted. Suchnucleotides encoding mutant angiogenic growth factors can be readilyobtained by site-directed mutagenesis (edit. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Section 8.1-8.5(1987)); gene amplification methods such as PCR (edit. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Section6.1-6.4 (1987); and general hybridization methods (J. Sambrook et al.,Molecular Cloning 2 nd ed., Cold Spring Harbor Press, Section 9.47-9.58(1989); edit. Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, Section 6.3-6.4 (1987)). Alternatively, the genes orfragments thereof may be chemically constructed based on their sequenceinformation.

A stringent condition for hybridization normally includes a washcondition of “1×SSC, 37° C.”. A more stringent condition would be a washcondition of “0.5×SSC, 0.1% SDS, 42° C.”, and a much more stringentcondition would be “0.1×SSC, 0.1% SDS 65° C.”. The more stringent thecondition, the higher the homology of the obtained polynucleotide to theprobe sequence. However, the hybridization conditions mentioned aboveare merely examples, and it should be understood that those skilled inthe art can select an appropriate condition for hybridization, takingthe nucleotide sequence, concentration and length of the probe; reactiontime; reaction temperature; concentration of the reagent; etc. intoconsideration.

The gene encoding an angiogenic growth factor of the present inventionisolated by the above-mentioned hybridization techniques normallyencodes a polypeptide that is highly homologous at the amino acidsequence level to the natural occurring angiogenic growth factor used asthe probe. “Highly homologous” herein refers to an identity higher than50%, preferably 65%, more preferably 75%, even more preferably 80%, muchmore preferably 90% and most preferably 95% or higher. Methods fordetermining sequence homology between polynucleotides are known in theart, and may be determined following the BLAST search algorithm (Karlinand Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993)).

A mutation of proteins may occur in nature too. As mentioned above,various isoforms of respective angiogenic growth factors are known inthe art. Such isoforms are also included in the angiogenic growthfactor-encoding gene to be used in the present invention, so long asthey retain the angiogenic activity of the native protein. It is wellknown that a protein modified by substitution, deletion, addition and/orinsertion of one or more amino acid residues in the sequence of aprotein can retain its original biological activity (G.Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA 79: 6409-6413(1982)).

To conserve the angiogenic activity of an angiogenic growth factor, itis preferable to mutate the amino acid residue into one that allows theproperties of the amino acid side-chain to be conserved. The propertiesof amino acids are generally classified into: (1) Hydrophobic aminoacids (alanine, isoleucine, leucine, methionine, phenylalanine, proline,tryptophane, tyrosine and valine)

(2) hydrophilic amino acids (arginine, asparagines, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serineand threonine); (3) amino acids having aliphatic side-chain (alanine,glycine, isoleucine, leucine, phenylalanine and valine); (4) amino acidshaving hydroxyl group-containing side-chain (serine, threonine andtyrosine); (5) amino acids having sulfur atom-containing side chain(cysteine and methionine); (6) amino acids having carboxylic acid- andamide-containing side chain-(aspargine, aspartic acid, glutamic acid andglutamine); (7) amino acids having base-containing side chain (arginine,histidine and lysine); and (8) amino acids having aromatic-containingside chain (histidine, phenylalanine, tyrosine and tryptophane).

Examples of proteins or polypeptides having one or more amino acidresidues added thereto include, but are not limited to, fusion proteins.For example, to prepare a polynucleotide encoding a fusion protein, afirst DNA encoding an angiogenic growth factor and a second DNA encodinganother protein or polypeptide are linked in frame. The protein orpolypeptide that can be fused to the angiogenic growth factor is notlimited to any specific protein or polypeptide.

The activity of a mutant protein can be confirmed according toconventional methods, using well-known assays. For example, theangiogenic activity of mutated proteins and polypeptides of angiogenicgrowth factors can be confirmed according to a method described in theExamples below, wherein the effect of a protein to induce therapeuticangiogenesis in rat ischemic hindlimb model is detected. Alternatively,the activity of a mutant NOS can be measured according to the methoddescribed in WO97/07824, wherein the activity of a protein to induceproliferation of hemangioendothelial cells is detected.

According to the present invention, a gene encoding a first angiogenicgrowth factor can be used alone or in combination with one or more genesencoding other angiogenic growth factors. Furthermore, genes encodingtranscriptional factors regulating the expression of angiogenic growthfactors, such as hypoxia-inducible factor (HIF)-1α and Ets-1, can bealso used in combination with the gene encoding an angiogenic growthfactor in the present invention.

The gene encoding an angiogenic growth factor and other genes usedaccording to needs in combination with the angiogenic growth factorencoding gene in the present invention are preferably inserted into (a)vector(s) which ensures expression of the genes in vivo, and which maybe administered to the patient by the “naked” DNA method into lesions orsurrounding muscle sites thereof. To express the gene(s), any expressionvector may be used, so long as it enables expression of the objectgene(s) in vivo. For example, such expression vectors include, but arenot limited to, PCAGGS (Gene 108: 193-200 (1991)), PBK-CMV (Stratagene),pcDNA3.1 (Invitrogen), pZeoSV (Invitrogen), etc. The expression vectormay further comprise regulatory genes, such as a promoter, enhancerand/or terminator, that are required for the expression of theangiogenic growth factor gene.

The expression vector(s) comprising an angiogenic growth factor gene maybe formulated as a pharmaceutical composition suitable for gene therapyby the naked DNA method. For example, for administration by injection,the vector comprising the gene is dissolved in an appropriate solution.Then, the solution comprising the vector is sterilized by filtrationaccording to particular needs and may be filled in an aseptic ampouleand such. According to needs, conventionally used carriers may be addedto the solution for injection.

Preferred solutions for dissolving the angiogenic growth factor-encodinggene include, buffer solutions, such as phosphate buffered saline (PBS),physiological saline, sucrose solution, glucose solution, sterilizedwater, etc. The experimental results reported herein suggest anddemonstrate that high transfection efficiency may be achieved by salineas well as PBS. Furthermore, higher transfection efficiency was obtainedwith the use of glucose solution as compared to sucrose solution. Thus,particularly preferred solutions for the present invention includesaline, PBS and glucose solution.

According to the present invention, an angiogenesis-dependent symptommay be treated or prevented in a subject by injecting into a muscle asuitable naked plasmid DNA under a condition wherein the pressure insidethe muscle is increased. Increasing “the pressure inside the muscle”means that the pressure on the surface of the cells of the muscle isincreased. Such increase in pressure can be achieved by injecting largevolumes of solutions, i.e., injecting the naked plasmid DNA dissolved ina solution of a large volume or together with an additional injection ofa solution. Alternatively, the additional solution, that without nakedplasmid DNA, can be injected after a sufficient time interval from theinjection of the naked plasmid DNA, so long as such results in anincrease in transfection efficiency.

Furthermore, according to the present invention, the transfectionefficiency of a naked plasmid DNA introduced into a subject can be alsoincreased by performing hyperbaric oxygen (HBO) therapy in combinationwith administration of the naked plasmid DNA. HBO therapy involvesexposing a subject to compressed oxygen (more than 1 atm., generally 3to several atm.). According to the invention, it is preferable toconduct the HBO therapy by exposing the subject to 100% oxygen.

For HBO therapy, a monoplace chamber (adapted for one person) compressedwith pure oxygen can be used; alternatively, the subject may be made tobreathe oxygen through a mask, headtent (oxygen tent) or endotrachealtube in a multiplace chamber with compressed air. HBO therapy is knownto increase the oxygen level in plasma, organs and tissues.

HBO therapy may be performed together with the intramuscular injectionof naked plasmid DNA, though it is preferable to start the therapyimmediately after the injection.

Although the dosage of the angiogenic growth factor-encoding gene variesdepending on the weight, age, sex and symptom of the patient, the kindof gene to be administered, the administration method and so on, oneskilled in the art can readily select an appropriate dose of the genefor therapeutic or preventive treatment of angiogenesis-dependentsymptoms using routine calculations and well-known algorithms.Generally, the gene is administered to an adult (calculated as a bodyweight of 60 kg) once every few days or few months at a range of 0.0001to 100 mg and preferably 0.001 to 10 mg. For administration to otheranimals, the amount of the gene may be converted for the amount per 60kg body weight can be administered.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended to otherwise limit the scope of the inventionin any way.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Any patents, patent applications andpublications cited herein are incorporated by reference.

EXAMPLE 1 General Methods

(1) In Vivo Gene Transfer Using Direct Intramuscular Injection Approach

Sprague-Dawley rats (400-500 g; Charles River Breeding Laboratories)were anesthetized with an intraperitoneal injection of sodiumpentobarbital (0.1 ml/100 mg). Naked luciferase gene (500 μl/animal) orcontrol (500 μg/animal) vector was carefully injected directly into thecenter of the pretibial muscle of the right hindlimb of rats with a 27 Gneedle (Terumo, Atsugi, Japan) (Y. Taniyama et al., Gene Ther. 8:181-189 (2000); M. Aoki et al., Gene Ther. 7: 417-427 (2000); Y.Taniyama et al., Circulation 104: 2344-2350 (2001); R. Morishita et al.,Circulation 105: 1491-1496 (2002)). A luciferase gene expression vectordriven by SV40 promoter (Promega Corporation, Madison, Wis.) was used asthe naked luciferase gene vector.

1) To increase the pressure within the muscle, the manchette ofsphygmomanometer was wrapped on the muscle injected with nakedluciferase plasmid DNA immediately after transfection.

2) To add pressure at the injection sites, additional intramuscularinjection of PBS solution without plasmid DNA was given to the muscleinjected with naked luciferase plasmid DNA at 0.5 or 5 hours aftertransfection.

(2) Analysis of Luciferase Activity

Firefly luciferase activity was measured using luciferase assay system(PicaGene™; Toyo-Inki, Tokyo, Japan). Rats were sacrificed 2 days aftertransfection of the luciferase gene by direct injection of naked plasmidinto the hindlimb. Tissue samples (200 mg around the injection site)were rapidly frozen in liquid nitrogen, and homogenized in lysis buffer.The tissue lysates were briefly centrifuged (3000 rpm, 10 min), and 20μl of supernatant was mixed with 100 μl of luciferase assay reagents.Measurement of the luminescent reaction was started 5 sec after theaddition of sample. Counting lasted for 10 sec, and the count in 10 secwas used as an index of luciferase activity (M. Aoki et al., J. Mol.Cell. Cardiol. 29: 949-959 (1997)).

(3) HBO Therapy (O₂ Exposures)

Rats were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg).Intramuscular injection of plasmid DNA was performed as described above.On the morning of exposure, animals were placed in a hyperbaric chamber.The chamber was flushed with 100% O₂ for 1.5 min to rise the O₂ levelto >99%. The animals were exposed to 2 atm 100% O₂ for 1 hour,immediately after the transfection.

(4) Statistical Analysis

All values are expressed as mean ±SEM. Analysis of variance withsubsequent Duncan's test was used to determine the significance ofdifferences in multiple comparisons. Differences with a P value lessthan 0.05 were considered significant.

EXAMPLE 2 Comparison of Transfection Efficiency into Rat Muscle In Vivo

Initially, we examined the effect of the injection volume on thetransfection efficiency of naked plasmid DNA comprising the luciferasegene. As expected, the activity of luciferase increased due to theplasmid DNA in a dose-dependent manner (FIG. 1; p<0.01) Interestingly,as shown in FIG. 1, the transfection of naked plasmid DNA increased inrelation with the increase in the injection volume of solution(PBS)(p<0.01). The increase in injection volume (100 μl at one site),rather than separate injections (25 ml at 4 sites or 12.5 ml at 8sites), gave high transfection efficiency (FIG. 2, p<0.01). Thus, thetransfection efficiency of naked plasmid DNA seemed to be related to theosmotic pressure.

To clarify this hypothesis, the inventors used the manchette ofsphygmomanometer on the hindlimb after injection, to increase thepressure from outside. Unexpectedly, neither the manchette-mediatedpressures at 150 and 300 mmHg increased the transfection efficiency(FIG. 3). Furthermore, the transfection efficiency was not affected byrepeated press of manchette (FIG. 3).

Thus, to increase the inside pressure, PBS without plasmid DNA wasintramuscularly injected on the same site of plasmid DNA transfection.As shown in FIG. 4 a, additive injection of PBS 30 minutes (0.5 hrs)after the first injection of plasmid DNA increased the luciferaseactivity (p<0.011). Nevertheless, similar additive injection of PBSalone 5 hours after the initial transfection did not increase theluciferase activity. Using 800 μg of plasmid DNA, similar results wereobtained (FIG. 4 b). In contrast, changes in the injection speed did notaffect the transfection efficiency (data not shown).

For further confirmation, HBO therapy was employed. In HBO therapy,animals are exposed to an environment of pure oxygen under highpressure. HBO therapy at 2 atm for 1 hour achieved a significantincrease in the luciferase activity in both animals injected withinjection volumes of 100 μl and 300 μl (FIG. 5, p<0.01). These resultsdemonstrated that the transfection efficiency of intramuscular injectionof naked plasmid DNA was dependent on the pressure at the cell surface.

Alternatively, an increase in osmotic pressure may affect thetransfection efficiency. Thus, the influence of the use of varioussolutions as injection vehicles of plasmid DNAs on transfectionefficiency was examined. As shown in FIG. 6 a, saline as well as PBSdemonstrated high transfection efficiency as compared to other buffers.Unexpectedly, the use of water as the injection vehicle diminished theluciferase activity. To increase osmotic pressure in vivo, glucose andsucrose solutions were also tested for their effect. Both sucrose andglucose solutions increased the expression of luciferase, and sucrosesolution rather than glucose solution significantly increased theluciferase activity as compared to water (p<0.01). However, the use of a30% sucrose solution caused injury at the injected site of the musclethus is not particularly preferred.

INDUSTRIAL APPLICABILITY

The present invention provides modified methods of plasmid DNA-basedgene delivery into the skeletal muscle that are safer and achieve highertransfection efficiency as compared to conventional methods.Specifically, the present invention provides a method for treating orpreventing diseases by intramuscular injection of suitable naked plasmidDNA under increased pressure inside the muscle. Furthermore, the presentmethod provides a method for treating or preventing diseases byintramuscular injection of suitable naked plasmid DNA in combinationwith hyperbaric oxygen (HBO) therapy.

According to the present methods, the amount of plasmid DNA to beadministered can be decreased and thus the potential cost for nakedplasmid DNA therapy can be reduced. Furthermore, these methods achieveefficient transfection without a viral vector, such as adenoviralvectors. In particular, the present methods are more safe as compared tomethods utilizing viral vectors and open up the possibility of genetherapy for a wide variety of diseases. Moreover, the combination ofnaked plasmid DNA administration and HBO therapy of the presentinvention may expand the utility of angiogenic growth factors in humanclinical gene therapy of angiogenesis-dependent conditions, such aswound healing, inflammatory disease, ischemic heart diseases, myocardialinfarction and peripheral arterial diseases.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method for treating or preventing an angiogenesis-dependent symptomcomprising the step of injecting a naked plasmid DNA encoding anangiogenic growth factor into a muscle under a condition wherein thepressure inside the muscle is increased.
 2. The method of claim 1,wherein the pressure inside the muscle is increased by adopting a largeinjection volume.
 3. The method of claim 1, wherein the pressure insidethe muscle is increased by injecting PBS after plasmid DNAadministration.
 4. The method of claim 1, wherein the naked plasmid DNAis diluted in saline, PBS, sucrose solution, or glucose solution.
 5. Themethod of claim 1, wherein the angiogenic growth factor is selected fromthe group consisting of: hepatocyte growth factor (HGF); vascularendothelial growth factor (VEGF); fibroblast growth factor (FGF); andnitric oxide synthase.
 6. The method of claim 5, wherein the nitricoxide synthase is selected from the group consisting of macrophagederived nitric oxide synthase, inducible nitric oxide synthase, andbrain derived nitric oxide synthase.
 7. The method of claim 1, whereinthe angiogenesis-dependent symptom is selected from the group consistingof: wounds, including bedsore and skin ulcer; inflammatory diseases;critical limb ischemia; ischemic heart diseases, including myocardialinfarction, angina pectoris, and heart failure; cerebral infarction;diabetic neuropathy; and spinal canal stenosis.
 8. A method for treatingor preventing angiogenesis-dependent symptom comprising the step ofadministering a naked plasmid DNA encoding an angiogenic growth factorinto a muscle in combination with hyperbaric oxygen (HBO) therapy. 9.The method of claim 8, wherein the HBO therapy is conducted by exposureof 100% oxygen.
 10. The method of claim 8, wherein the subject to betreated is subjected to the HBO therapy immediately after the plasmidDNA administration.
 11. The method of claim 8, wherein the naked plasmidDNA is diluted in saline, PBS, sucrose solution, or glucose solution.12. The method of claim 8, wherein the angiogenic growth factor isselected from the group consisting of: hepatocyte growth factor (HGF);vascular endothelial growth factor (VEGF); fibroblast growth factor(FGF); and nitric oxide synthase.
 13. The method of claim 12, whereinthe nitric oxide synthase is selected from the group consisting ofmacrophage derived nitric oxide synthase, inducible nitric oxidesynthase, and brain derived nitric oxide synthase.
 14. The method ofclaim 8, wherein the angiogenesis-dependent symptom is selected from thegroup consisting of: wounds, including bedsore and skin ulcer;inflammatory diseases; critical limb ischemia; ischemic heart diseases,including myocardial infarction, angina pectoris, and heart failure;cerebral infarction; diabetic neuropathy; and spinal canal stenosis.